The loss of bone mass and quality is a serious health problem which is even more common in elderly patients.
The success in the regeneration of a bone defect using three-dimensional materials, which are initially colonized by progenitor cells in vitro, depends to a great extent on the characteristics and structure of the material.
Biomaterials have been used for almost a century to repair or replace bone segments of the musculoskeletal system.
The use of autogenous bone grafts, i.e., from the individual himself, is a widely used method for filling bone cavities and for surgical reconstructions. However, there is a limited bone supply and the patient must furthermore be subjected to additional trauma in order to obtain the graft. Another option is donor allografts which also have drawbacks such as a slower neoformation rate, lower osteogenic capacity, reabsorption rate, lower revascularization as well as a higher risk of immunogenic response and transmission of pathogenic agents.
It is ideal to obtain a material similar to bone, which is biocompatible, does not present adverse biological reactions, is reabsorbable and is gradually degraded as the new tissue is formed, thus progressively transferring the loads to the new bone, preventing a second surgical intervention for removing the implant. A material the degradation products of which are easy to eliminate and non-toxic, which is osteoinductive and induces bone tissue formation is also ideal.
In the organism, bone degradation and reabsorption are carried out by osteoclasts. They are cells derived from monocytes which are fixed to the surface of the bone. Once fixed, they start releasing protons to the exterior, for the purpose of lowering the pH of the external medium. With this acidic environment, the hydroxyapatite crystals forming part of the mineral component of bone are solubilized. The hydroxyapatite of bone is solubilized in amorphous calcium phosphate particles, which are eliminated by macrophages, or in Ca2+ and PO43− ions which accumulate in the extracellular fluid. These ions diffuse towards the blood capillaries, entering the systemic circulation to be eliminated by urine through the kidney. These released ions can also be reused by osteoblasts to form new bone. Osteoclasts are also in charge of the degradation of the organic phase of bone by means of enzymatic processes.
The research in new biomaterials for bone repair attempts to reduce the need for bone grafts as much as possible, seeking an artificial substitute which over time is reabsorbed and/or is integrated with the adjacent bone and furthermore serves as fixing in osteoporotic fractures. The mechanical properties of the bone substitute must be as similar as possible to those of spongy bone. The material must furthermore aid in the stability of the fracture and be resistant enough to decrease the necessary external support or immobilization time. Said material must be reabsorbable, biocompatible and osteoinductive, i.e., it must attract mesenchymal cells and other cell types located close to the implant and favor the differentiation thereof into osteoblasts, and also osteoconductive, i.e., it mist act as a mold for the formation of new bone.
Seeking a similarity with what occurs in the organism, the non-reabsorbable materials used up until now are being substituted in bone implants with reabsorbable materials. These biomaterials do not interfere in the development and growth of the new bone formed, since they are gradually replaced by host tissue. Furthermore, they have a higher biocompatibility, they participate naturally in bone reconstruction and it is not necessary to remove them by means of surgery, after bone regeneration. These materials have to remain for the sufficient time for correct bone regeneration to take place and disintegrate gradually without harming the patient and without intervening in the correct development and growth of the bone.
The biomaterials which set forming a mineral calcium phosphate are especially interesting in bone regeneration since they resemble the mineral phase of natural bone and are susceptible of bone remodeling and of reabsorption due to their metastable crystal structure.
The reabsorbable materials which are being used as bone substitutes include calcium phosphates; hydroxyapatite (HAP), tricalcium phosphate (B-TCP) and dicalcium phosphate dihydrate (DCPD) (Stubbs et al., 2004; Schnettler et al., 2004). These materials have an excellent biocompatibility due to their chemical and crystalline similarity to the mineral component of bone, but have difficulties in relation to solubility and reabsorption capacity in vivo.
Hydroxyapatite (HAP) has been one of those which has aroused the greatest interest. This material is per se the inorganic phase from which bones are formed and it has therefore been widely used in bone regeneration. An example of this are some commercial products such as Interpore 200® Interpore 500®, Cerasorb® and Collagraft®. However, and due to the fact that it has one of the most stable crystal structures, the material has a slow reabsorption.
HAP is the material having the highest biocompatibility, as it is the most similar one to the crystals formed by bone, but it is not reabsorbable in vivo. The degradation of this material occurs by contact with solutions with a low pH and by phagocytosis. By means of dissolution the amorphous calcium phosphate particles are released, and can be eliminated by macrophages by phagocytosis or be embedded in the new bone formed. Macrophages can dissolve these particles and restore Ca and P to the pool of the organism (Frayssinet et al., 1999; Benahmed et al., 1996). However, it has not been observed that these particles give rise to osteoclast activation (Frayssinet et al., 1999).
All the studies conducted corroborate the resistance of this material to degradation once it is implanted in the organism, due to its poor solubility at physiological pHs. Implants of this type in animals are reabsorbed by 5.4% in 6 months compared to those based on B-TCP, which are reabsorbed by 85%. (Eggli et al., 1988).
In humans, the implants made with Bio-Oss (HAP) are considered as non-reabsorbable, since the studies conducted demonstrate that between 3-6 years are needed for them to be reabsorbed due to osteoclast activity (Taylor et al., 2002). The presence of this material in the organism for so much time can interfere in the bone remodeling process, as well as in the osseointegration capacity (Affe et al., 2005; De Boever 2005).
As a result, this material has traditionally been used in mixtures with organic material as polymers to increase the reabsorption thereof. Examples of these applications are described in U.S. Pat. No. 5,866,155, which describes the incorporation of hydroxyapatite in polylactic matrices, or in U.S. Pat. No. 5,741,329, which is a variation of U.S. Pat. No. 5,866,155 which intends to correct several defects derived from the local acidification of the medium after incorporating cements in the organism.
To that end, for the purpose of improving the capacity of reabsorption of calcium phosphates and increasing their osteoconductive capacity, crystalline calcium phosphate phases less stable than hydroxyapatite 6, such as B-TCP and DCPD (Brushite), having better solubility and reabsorption in vivo, have been used in recent years.
B-TCP has more osteoconductivity and a better reabsorption than HAP (Franco et al., 2006). It is considered as a moderately reabsorbable material, in in vivo studies it has been observed that at least one year is needed for its reabsorption in animals and from 6 to 8 months in humans (Wiltfang et al., 2003; Suba et al., 2004). Its degradation increases calcium deposits and this is associated with a higher alkaline phosphatase activity, which enzyme is involved in bone formation (Trisi et al., 2003; Sugawara et al., 2004).
DCPD is also biocompatible, osteoconductive and the most reabsorbable due to being the most soluble at physiological pHs. This allows new bone to be formed more quickly. It is biodegraded in physiological environments and it is reabsorbed by adjacent cells (Tris et al., 2003). It is proved to be reabsorbed in vivo up to three times quicker than HAP and B-TCP (Herron et al., 2003; Chow et al., 2003; Tas & Bhaduri 2004; Tamini et al., 2006).
Studies suggest that part of the DPDC material can be converted into HAP after its implantation, which can delay the elimination of the implant by osteoclasts by several weeks (Constanz et al., 1998). This conversion can make the cells acidify the medium and make the biocompatibility of the material decrease together with a reduction in its reabsorption. The addition of Mg and Ca (calcium carbonate) salts or the combination thereof with BTCP can prevent this conversion.
Using this material it is observed that generation of bone and elimination of the material occur in a balanced manner after the 4th week (Fallet et al., 2006) and the 8th week post-intervention (Constanz et al., 1998). This is important because if the degradation were greater than the synthesis instability and inflammatory reactions would be created.
Thus, among these calcium phosphates, brushite (DPCD) is one of the materials of greatest interest in bone regeneration. Due to the interesting properties thereof, there are currently brushite cements designed for setting in situ. Thus, for example, U.S. Pat. No. 6,733,582 and US2006213398 claim brushite cements with in situ setting, Chronoss Inject® being an already marketed product of this type. However, this material has a great problem when it is sterilized since it decomposes when it is heated, which makes its appropriate sterilization difficult.
The state of the art contemplates different publications relating to the sterilization of cements which can be used as bone material substitutes, as well as about the methods used to make said matrices and their sterilization. However, as reflected in patent application JP2004018459, when said cements are sterilized by autoclave, the characteristics of said cements are altered, translating into obtaining bone mineral substitutes which do not meet the characteristics necessary for their use in bone regeneration in terms of reabsorption, stability and colonization and other essential properties.
As occurs with DPCD, Monetite is reabsorbed in vivo in a similar time and manner. It is gradually dissolved at physiological pHs in the extracellular tissues surrounding the implant and the actual cells colonizing it (endothelial cells, osteoclasts, osteoblasts, macrophages . . . ) would be responsible for the elimination or reuse thereof as occurs in bone.
Documents such as US20060263443 present Monetite, dicalcium phosphate anhydrous (DCPA), obtained by dehydration of Brushite, in combination with other calcium phosphate biomaterials. Due to the combination, the sterilization results were not acceptable for using these materials in implants and bone regeneration. Additionally, these materials are reaction intermediates and not structures with their own capacity to be used in the technical field of bone regeneration.
Additionally, for correct bone regeneration, it is necessary for the biomaterial to have a suitable porosity allowing cell colonization and proliferation, vascularization, increase of the surface of contact and therefore increase of the surface of interaction with the host tissue which allows the acceleration of bone regeneration. These characteristics must be accompanied by a correct reabsorption rate providing the cells with the time necessary for regeneration.
Thus, Gbureck, Uwe et al., 2007, relate to Brushite and Monetite implants prepared by means of the three-dimensional printing technique. To achieve said implants, matrices of brushite which are hydrothermally dehydrated, being transformed into Monetite, are obtained first. However, Table 2 of said article shows that the calcium phosphate material defined as Monetite in said article only has a Monetite content of 63%, not specifying the size or the distribution of its porosity, having a destructured porosity. Thus, said structures are not valid for the purposes of the present invention.
U.S. Pat No. 6605516 presents bone substitutes with a controlled anatomical shape which adjust exactly to the morphology of the injury. Said substitutes are formed by chemically consolidated calcium phosphate cement materials. The invention also relates to porogenic phases and molds which allow obtaining calcium phosphates with macroporous architectures and external geometries by means of using said molds. However, in its particular embodiments, the invention presents Brushite materials, not presenting Monetite materials and the macroporous structures presented therein also not being valid for the object of the present invention. Thus, the present invention provides matrices of monetite (metastable calcium phosphate phase of monetite), with a high thermal stability which allows sterilizing the material by means of autoclaving, thus simplifying the sterilization processes and which, furthermore, due to its specific structural arrangement of pores, which arrangement is obtained as a result of a specific design of the material, involves an improvement of the osteoinductive capacity of materials proposed by the state of the art since it is synthesized in the form of a porous block with defined structured macroporosity characteristics, increasing the specific surface area, as well as the area of contact with the osteoblasts and facilitating the nutrient transport processes for cells, a crucial factor for bone generation, all of this together with the high capacity of reabsorption thereof in the suitable time period for the adjacent cells to colonize the material and be able to replace the reabsorbed material with physiological bone matrix.
The in vitro degradation of the matrices of the invention does not affect cell proliferation and they are furthermore bioactive, non-cytotoxic, non-mutagenic and hemocompatible.